§Should be performed whenever the electrophoretic or HPLC is characteristic of HbS

§Also whenever there is a variant haemoglobin of uncertain significance as there are a number of variants that the HbS glutamic acid for valine substitution and others that change its electrophortic properties but still mean that the patient is prone to sickling.

§Not useful when there are low levels of the variant haemoglobin (will only detect down to 20%) – need to correct the HCT to 50% when the patient is anaemic to avoid false negatives.

Technique

§Packed red cells reconstitute to a haematocrit of 50%.

§+ PO4 buffer containing reducing and lysis agents

§Sickle Hb is induced to sickle by the reducing agent and gets trapped in red cells

§Normal Hb is lysed

§Centrifuge and read

§Any sickle Hb results in turbidity

§False negatives (limit of detection 20%)

§Babies <6months

§Massively transfused patients

§False positives occur with

§Hyperlipidaemia

§Paraprotein

§Heinz body haemolytic anaemia

§Very high white count or nucleated red cell count

Haemoglobin electrophoresis

§Best performed on lysed packed red cells

§Very common technique for initial detection of variant haemoglobin though HPLC is increasingly taking its place.

§Alkaline electrophoresis – pH 8.2-8.6

§Provisional ID of (in this order away from the origin (ie. C migrates the least distance):